Low Expression Of PTEN In CCRCC Contributes To Chemoresistance Through Activating Akt/HDM2Signaling Pathway

Objective: To investigate roles of low expression of PTEN in CCRCC chemoresistance,and to elucidate its possible mechanisms.Methods:Western blotting and immunochemistry were used to determine theexpression of tumor suppressor gene PTEN in CCRCC; PTEN knockdown, together withflow cytometry analysis or CCK8analysis, was used to study the effects of PTEN silence onCCRCC cell apoptosis and cell proliferation ability, respectively; PTEN knockdown,together with immunoprecipitation and western blotting, was used to investigate the effectsof PTEN on etoposide induced cell apoptosis and the related molecular mechanisms.Results: PTEN mRNA and protein expression levels were significantly decreased inCCRCC tissues compared with the adjacent non-neoplastic renal samples. After transfectionwith PTEN shRNA, PTEN mRNA and preotein expression levels were inhibited efficientlyin ACHN cells, apoptosis induced by etoposide was reduced significantly, and cellproliferation ability was enhanced, which indicated inhibition of PTEN led to apoptosisresistance, and promotion of cell proliferation; After treating with etoposide, PTEN proteinexpression levels did not show any obvious changes, which illustrated etoposide did noteffect on PTEN directly. P-Akt, p-HDM2and HDM2erpression levels in ACHN cellstreated with etoposide were reduced; Meanwhile, p53, PUMA and Cleaved PARP erpressionlevels were increased, all this pointed out that etoposide inhibited Akt signaling pathway, andmight induced cell apoptosis through a p53-dependent pathway; On the opposite, afterknocking down of PTEN in ACHN cells, p-Akt, p-HDM2and HDM2erpression levels wereincreased, accompanying with decreased expression of p53, PUMA and Cleaved PARP, thisshowed us that inhibition of Akt signaling induced by etoposide was released by PTENinhibition, and simultaneously p53-dependent apoptosis was restrained. The results ofwestern blotting revealed that p-Akt, p-HDM2and HDM2erpression levels were increasedin CCRCC tissue with decreased p53expression, and immunochemistry experiments furtherconfirmed the results; Immunoprecitation experiments results showed that amount of p53which interacts with HDM2was decreased, and it was increased after PTEN knocking out.In CCRCC tissue, the amount of p53which interacts with HDM2was also increaseddefinitely. Together with the above results, we can conclude that the interaction between HDM2and p53was inhibited by etoposide, which abolished the degradation of p53inducedby HDM2; And this results was reversed by PTEN inhibition.Conclusion: Akt/HDM2signaling pathway was constitutively activated in CCRCCtissues, which was concomitant with low p53expression level. Silence of PTEN in CCRCCcells resulted inthe inhibition of apoptosis induced by etoposide, as well as the enhancedproliferation ability. Meanwhile, Akt/HDM2signaling pathway was activated, interactionbetween HDM2and p53was increased and the level of p53protein was lowered, whichinhibited the activity of p53. All in all, low expression of PTEN activates Akt/HDM2signaling pathway in CCRCC, and contributes to chemoresistance through inhibitingp53-dependent apoptosis.